The present invention relates generally to the fields of chemistry and biology. More particularly, the present invention relates to compositions and methods for use in measuring gene expression.
A reporter gene assay measures the activity of a gene""s promoter. It takes advantage of molecular biology techniques, which allow one to put heterologous genes under the control of any promoter and introduce the construct into the genome of a mammalian cell [Gorman, C. M. et al., Mol. Cell Biol. 2: 1044-1051 (1982); Alam, J. and Cook, J. L., Anal. Biochem. 188: 245-254, (1990)]. Activation of the promoter induces the reporter gene as well as or instead of the endogenous gene. By design the reporter gene codes for a protein that can easily be detected and measured. Commonly it is an enzyme that converts a commercially available substrate into a product. This conversion is conveniently followed by either chromatography or direct optical measurement and allows for the quantification of the amount of enzyme produced.
Reporter genes are commercially available on a variety of plasmids for the study of gene regulation in a large variety of organisms [Alam and Cook, supra]. Promoters of interest can be inserted into multiple cloning sites provided for this purpose in front of the reporter gene on the plasmid [Rosenthal, N., Methods Enzymol. 152: 704-720 (1987); Shiau, A. and Smith, J. M., Gene 67: 295-299 (1988)]. Standard techniques are used to introduce these genes into a cell type or whole organism [e.g., as described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Expression of cloned genes in cultured mammalian cells. In: Molecular Cloning, edited by Nolan, C. New York: Cold Spring Harbor Laboratory Press, 1989]. Resistance markers provided on the plasmid can then be used to select for successfully transfected cells.
Ease of use and the large signal amplification make this technique increasingly popular in the study of gene regulation. Every step in the cascade DNAxe2x86x92RNAxe2x86x92Enzymexe2x86x92Productxe2x86x92Signal amplifies the next one in the sequence. The further down in the cascade one measures, the more signal one obtains.
In an ideal reporter gene assay, the reporter gene under the control of the promoter of interest is transfected into cells, either transiently or stably. Receptor activation leads to a change in enzyme levels via transcriptional and translational events. The amount of enzyme present can be measured via its enzymatic action on a substrate. The substrate is a small uncharged molecule that, when added to the extracellular solution, can penetrate the plasma membrane to encounter the enzyme. A charged molecule can also be employed, but the charges need to be masked by groups that will be cleaved by endogenous cellular enzymes (e.g., esters cleaved by cytoplasmic esterases).
For a variety of reasons, the use of substrates which exhibit changes in their fluorescence spectra upon interaction with an enzyme are particularly desirable. In some assays, the fluorogenic substrate is converted to a fluorescent product. Alternatively, the fluorescent substrate changes fluorescence properties upon conversion at the reporter enzyme. The product should be very fluorescent to obtain maximal signal, and very polar, to stay trapped inside the cell.
To achieve the highest possible sensitivity in a reporter assay one has to maximize the amount of signal generated by a single reporter enzyme. An optimal enzyme will convert 105 substrate molecules per second under saturating conditions [Stryer, L. Introduction to enzymes. In: Biochemistry, New York: W. H. Freeman and company, 1981, pp. 103-134]. xcex2-Lactamases will cleave about 103 molecules of their favorite substrates per second [Chang, Y. H. et al., Proc.Natl.Acad.Sci.USA 87: 2823-2827 (1990)]. Using a fluorogenic substrate one can obtain up to 106 photons per fluorescent product produced, depending on the type of dye used, when exciting with light of the appropriate wavelength. The signal terminates with the bleaching of the fluorophore [Tsien, R. Y. and Waggoner, A. S. Fluorophores for confocal microscopy: Photophysics and photochemistry. In: Handbook of Biological Confocal Microscopy, edited by Pawley, J. B. Plenum Publishing Corporation, 1990, pp. 169-178]. These numbers illustrate the theoretical magnitude of signal obtainable in this type of measurement. In practice a minute fraction of the photons generated will be detected, but this holds true for fluorescence, bioluminescence or chemiluminescence. A good fluorogenic substrate for a reporter enzyme has to have a high turnover at the enzyme in addition to good optical properties such as high extinction and high fluorescence quantum yield.
It is an object of the present invention to provide xcex2-lactamase substrate compounds. It is a further object of the invention to provide membrane-permeant compounds. The membrane-permeant compounds may be transformed into substantially membrane-impermeant compounds.
Another object of the invention is to provide xcex2-lactamase reporter genes. A further object of the present invention is to create cells containing the xcex2-lactamase reporter genes functionally linked to a promoter such that when the promoter is turned on, the reporter gene will be expressed. Expression of the xcex2-lactamase is measured with the xcex2-lactamase substrates which emit light after hydrolysis by the xcex2-lactamase.
A further object of the invention is to use the xcex2-lactamase reporter genes in cells and the xcex2-lactamase substrate compounds of the present invention to screen for biochemical activity.
In accordance with the present invention, fluorogenic substrates are provided of the general formula I 
wherein:
one of X and Y is a fluorescent donor moiety or a membrane-permeant derivative thereof, and the other is a quencher moiety, an acceptor fluorophore moiety or a membrane-permeant derivative thereof;
Rxe2x80x2 is selected from the group consisting of H, lower alkyl, (CH2)nOH, (CH2)nCOORxe2x80x3, and xe2x95x90NOJ, in which n is 0 or an integer from 1 to 5 and J is H, Me, CH2COOH, CHMeCOOH, and CMe2COOH;
Rxe2x80x3 is selected from the group consisting of H, physiologically acceptable metal and ammonium cations, xe2x80x94CHR2OCO(CH2)nCH3, xe2x80x94CHR2OCOC(CH3)3, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, betamorpholinoethyl, dialkylaminoethyl, dialkylaminocarbonyloxymethyl, in which R2 is selected from the group consisting of H and lower alkyl;
A is selected from the group consisting of S, O, SO, SO2 and CH2;
Zxe2x80x2 is a linker for X; and
Zxe2x80x3 is a linker for Y.
In another aspect, this invention provides methods for determining whether a sample contains xcex2-lactamase activity. The methods involve contacting the sample with a compound substrate of the invention, which exhibits fluorescence resonance energy transfer when the compound is excited; exciting the compound; and determining the degree of fluorescence resonance energy transfer in the sample. A degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of xcex2-lactamase activity. One embodiment of this method is for determining the amount of an enzyme in a sample. According to this method, determining the degree of fluorescence resonance energy transfer in the sample comprises determining the degree at a first and second time after contacting the sample with the substrate, and determining the difference in the degree of fluorescence resonance energy transfer. The difference in the degree of fluorescence resonance energy transfer reflects the amount of enzyme in the sample.
In another aspect, this invention provides recombinant nucleic acid molecule comprising expression control sequences adapted for function in a vertebrate cell and operably linked to a nucleotide sequence coding for the expression of a xcex2-lactamase. It also provides recombinant nucleic acid molecules comprising expression control sequences adapted for function in a eukaryotic cell and operably linked to a nucleotide sequence coding for the expression of a cytosolic xcex2-lactamase. In certain embodiments, the invention is directed to mammalian host cells transfected with these recombinant nucleic acid molecules.
In another aspect, this invention provides methods for determining the amount of xcex2-lactamase activity in a cell. The methods involve providing a host cell transfected with a recombinant nucleic acid molecule comprising expression control sequences operatively linked to nucleic acid sequences coding for the expression of a xcex2-lactamase; contacting a sample comprising the cell or an extract of the cell with a substrate for xcex2-lactamase; and determining the amount of substrate cleaved, whereby the amount of substrate cleaved is related to the amount of xcex2-lactamase activity.
In another aspect, this invention provides methods for monitoring the expression of a gene operably linked to a set of expression control sequences. The methods involve providing a host cell transfected with a recombinant nucleic acid molecule comprising expression control sequences operatively linked to nucleic acid sequences coding for the expression of a xcex2-lactamase, except if the eukaryote is a fungus, wherein the xcex2-lactamase is a cytosolic xcex2-lactamase; contacting a sample comprising the cell or an extract of the cell or conditioned medium with a substrate for xcex2-lactamase; and determining the amount of substrate cleaved. The amount of substrate cleaved is related to the amount of xcex2-lactamase activity.
In another aspect, this invention provides methods for determining whether a test compound alters the expression of a gene operably linked to a set of expression control sequences. The methods involve providing a cell transfected with a recombinant nucleic acid construct comprising the expression control sequences operably linked to nucleic acid sequences coding for the expression of a xcex2-lactamase except if the eukaryote is a fungus, wherein the xcex2-lactamase is a cytosolic xcex2-lactamase; contacting the cell with the test compound; contacting a sample comprising the cell or an extract of the cell with a xcex2-lactamase substrate; and determining the amount of substrate cleaved, whereby the amount of substrate cleaved is related to the amount of xcex2-lactamase activity. In one embodiment of the methods, the substrate is a compound of this invention. The step of determining the amount of substrate cleaved comprises exciting the compound; and determining the degree of fluorescence resonance energy transfer in the sample. A degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of xcex2-lactamase activity.
In another aspect, this invention provides methods of clonal selection comprising providing cells transfected with a recombinant nucleic acid molecule comprising the expression control sequences operably linked to nucleic acid sequences coding for the expression of a cytosolic xcex2-lactamase; contacting the cells with a substance that activates or inhibits the activation of the expression control sequences; contacting the cells with a compound of claim 9 which is converted into a substrate; determining whether substrate is cleaved within each individual cell, whereby cleavage reflects xcex2-lactamase activity; selecting and propagating those cells with a selected level of xcex2-lactamase activity. In a further embodiment, the method further involves culturing selected cells in the absence of activator for a time sufficient for cleaved substrate to be substantially lost from the cells and for xcex2-lactamase levels to return to unactivated levels; incubating the selected cells with a compound of claim 9 which is converted into a substrate; and selecting cells that have not substantially cleaved the substrate.